The members of the Transforming Growth Factor beta (TGFβ) family are cytokines involved in essential cellular functions such as proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis, immune response, cell adhesion, and also play a key role in pathophysiology of disease states as different as chronic inflammatory conditions and cancer. TGFβ initiates signaling by binding and bringing together type II (TβRII) and type I (TβRI) receptor serine/threonine kinases on the cell surface. TβRII phosphorylates the TβRI, which in turn phosphorylates receptor regulated SMADs (R-SMADs), i.e., SMAD2 and SMAD3. Activated SMADs heterodimerize with a common-partner SMAD4. Subsequently, the heterodimers, i.e., SMAD2/SMAD4 and SMAD3/SMAD4 translocate into the nucleus, where they cooperate with other transcription factors to modulate the expression of TGFβ target genes.
A negative feedback mechanism mediated by inhibitory Smads such as Smad6 and Smad7 acts to inactivate TGFβ signalling pathways by preventing the interaction of the TGFβ receptor complex with Smad2 and Smad3 (Ulloa et al. 1999; Kaysak et al. 2000; Massague et al. 2005). Moreover, the inhibitory Smad7 may recruit phosphatases and ubiquitin ligases to the activated TGFβ receptors and thereby inactivate said receptors by promoting their dephosphorylation and degradation.
Most cell types express three types of receptors for TGF-β. These are designated Type I (53 kDa), Type II (70-85 kDa) and type III (250-350 kDa). The Type III receptor, a proteoglycan that exists in membrane-bound and soluble forms, binds TGF-β1, TGF-β2 and TGF-β3 but does not appear to be involved in signal transduction. The Type II receptor is a membrane-bound serine/threonine kinase that binds TGF-β1 and TGF-β3 with high affinity and TGF-β2 with a much lower affinity. The Type I receptor is also a membrane-bound serine/threonine kinase that apparently requires the presence of the Type II receptor to bind TGF-β. Current evidence suggests that signal transduction requires the cytpolasmic domains of both the Type I and Type II receptors. A short form of TβRII has been described by the Whitehead Institute for Medical Research (WO9309228).
An alternatively spliced form of the Type II Receptor, referred to as TβRIIB, was described for mouse and human (Suzuki et al.,1995; Ogasa et al., 1996) and comprises an additional 75 bp coding for 25 amino acids. Thus, the alternative splicing results in an insertion of 26 amino acids in exchange for Val32 in the mouse and human extracellular domain of the receptor. This structural alteration leads to a new binding site for TGF-β2 without abolishing binding to the other isoforms, TGF-β1 and -β3. Both TβRII and TβRIIB bind TGF-β1 and TGF-β3 with high affinity. However, only TβRIIB also binds TGF-β2 with high affinity in the absence of TβRIII.
While TβRII is ubiquitously expressed, the splicing variant TβRIIB shows a restricted expression pattern in osteoblasts, mesenchymal precursor cells with upregulated levels during their differentiation into myoblasts, and in the heart. In the absence of TBRIII, TβRIIB binds to TGF-β2 and signals without the requirement of TβRIII. TβRIIB heterodimerizes with the wild-type or short form TβRII and binds all the three ligands TGF-β1, TGF-β2 and TGF-β3 (Krishnaveni et al., 2006). TβRIIB may play an important role in TGF-β2 binding and signaling in cells lacking TβRIII (Nikawa 1994; Rotzer, D. et al., 2001). In mammals the three TGF betas TGF-β1, TGF-β2, and TGF-β3 often show overlapping functions despite the fact that isoform specific knock-out mice revealed non-redundant and non-overlapping phenotypes.
Expression of the variant TβRIIB was found in all prostate cell lines studied with a preferential localization in epithelial cells in some human prostatic glands (Konrad et al., 2007). The expression of TβRIIB correlates with the unique expression pattern of TGF-β2 in chondrocytes and osteocytes (Rotzer et al., 2001). TGF-β2 is the only ligand that has a demonstrated role in epithelial mesenchymal cell transformation (EMT), a process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. In carcinoma cells, EMT can be associated with increased aggressiveness, and invasive and metastatic potential.
Previously performed research by Del Re et al. (2004) showed that the soluble extracellular domain of the TβRII, consisting of the extracellular domain of the receptor and the Fc part of a human immunoglobulin, bound TGF-β1 and TGF-β3 with high affinity but did not bind TGF-β2 in the same dose range.
While TGFβ has been considered a tumor suppressor factor because it promotes cell growth inhibition, apoptosis and differentiation (Gorelik and Flavell 2002), an extensive number of studies attest to the fact that TGFβ acts as a potent tumor promoter in established breast carcinoma, melanoma, gliomas among others.
In late stage tumor, breast cancer cells synthesize and secrete high levels of active TGFβ protein that can be found in both tumor cells and in plasma of breast cancer patients, both of which are associated with poor prognosis (Gorelik and Flavell 2002). As tumors progress, tumor-derived TGFβ becomes oncogenic by constitutively inducing epithelial to mesenchymal transition (EMT) and tumor associated angiogenesis and by suppressing tumor specific immunity, which combined promotes tumor growth and metastasis. As a pro-metastatic factor, TGFβ induces both the degradation of extracellular matrix and epithelial-to-mesenchymal transition of normal and transformed epithelial cells and thus enhanced migratory ability. In addition, TGFβ promotes myofibroblast differentiation and angiogenesis. Tumor derived-TGFβ also suppresses antitumor immune response by directly inhibiting the activation of cytolytic T cells, NK cells and macrophages, as well as interfering with dendritic cell function.
Consistent with this notion, several therapeutic approaches target TGFβ pathways for the treatment of invasive cancers such as breast cancer and melanoma. For instance, intracellular inhibition of TGFβ receptor I (TβRI) kinase with small-molecule inhibitors (Ki26894, SD-093 and SB-203580), effectively reduces number and size of lung metastases in both orthotropic xenografts and experimental metastasis models of breast carcinoma (Ge et al. 2006). Other small compounds (SD-093 and LY580276), inhibitors of epithelial-to-mesenchymal transition, also suppress tumor cell invasion and metastasis (Peng et al. 2005). In addition, antagonists of TGFβ binding to heteromeric receptor, such as a soluble Fc:TGFβ type II receptor fusion protein (Fc:TβRII), have shown significant reduction of tumor cell motility, intravasation, and lung metastases in three experimental models of breast cancer. However, this treatment strategy did not alter cellular proliferation (Muraoka et al. 2002), which indicates that the antimetastatic effect of Fc:TβRII in vivo was independent of tumor cell proliferation. Similar results were obtained with a monoclonal anti-TGFβ antibody (1D11), which also suppresses metastasis in highly metastatic model of breast cancer (4T1 cells), without significantly affecting tumor cell proliferation (Nam et al. 2006). Not only does TGFβ act as a prometastatic factor in advanced breast cancer, but it also exerts severe deleterious effects on several components of the immune response against cancer cells, abolishing the effector functions of macrophages, cytotoxic T cells, dendritic cells and NK cells, where TGFβ acts as a negative regulator of IFNγ production via its mediators SMAD2, SMAD3 and SMAD4. WO9804802 and US20050203022 disclose a fusion of the extracellular ectodomain of TβRII with the IgG immunoglobulin heavy chain. This molecule acts as a decoy trap for TGFβ.
Proinflammatory cytokines such as IL-2 constitute useful adjuvants for which extensive clinical experience exists for treatment of cancer. Cytokines can be used independently and combined as part of a fusokine to generate whole cell tumor vaccines as previously published (Stagg et al. 2004). Indeed, IL-2 is able to promote an innate antitumor response by inducing loco-regional tumor rejection, acting as an autocrine factor for T cells and supporting the development of cytotoxic T cells, and by stimulating NK cell proliferation and cytolytic activity. Despite the potent proinflammatory response initiated by cytokines, it has been recently discovered that tumor-derived TGFβ acts as a powerful and overwhelming dominant negative effect on the immune system, especially when a large tumor burden exists (Penafuerte and Galipeau 2008).